By David N. LeffScience Editor

It happened last week in New Orleans: A trio of superannuatedbacteria awoke from their multi-million-year hibernation to help resetthe molecular clock of evolution.

Microbiologist RaLl Cano reported to the 1996 annual meeting of theAmerican Society of Microbiology (ASM) on "The germination andgenome analysis of ancient spores in amber." He was addressingsome 160 attendees at a seminar on "The stress of prolongeddormancy: Maintaining genome integrity."

This ASM event was organized and co-chaired by microbiologistJohn Battista, of Louisiana State University in Baton Rouge. "Ipersonally brought RaLl in," Battista told BioWorld Today, "to bethe champ of prolonged microbial dormancy." He explained: "Therewere several people in the session who talked about organisms thatcould either be desiccated, or existed as spores, that you could reviveafter several hundred years. So one of my interests in having Canocome in and talk was to push the envelope a bit."

Cano chairs microbiology at California Polytechnic State University,in San Luis Obispo. "At the seminar," he told BioWorld Today, "Ilooked at the question: How can I tell that a microorganism isancient? Just because it is, does it have to be different from modernones? So we focused on a way of measuring microbial diversity ofmodern organisms."

Bacterial Shelf Life: Millions Of Years

He described three specimens of primordial Bacillus sphaericusspores, recovered from soil samples embedded in amber 25 million to40 million years ago, from the Dominican Republic and Mexico.

Taking painstaking precautions to prevent contamination bycontemporary DNA, Cano and his associates cracked the amberpieces, extracted the soil samples, ground up the tissue, grew therecovered bacteria in culture medium, then isolated and re-awakenedsurviving spores from the putative ancient bacillus specimens. (SeeBioWorld Today, Aug. 11, 1994, p. 1.)

"To identify the bacterial species, we did fatty-acid analysis of theirmembranes," he recounted, "and by comparing these among variousmembers of a single species, we could actually look at diversitybetween species, and define their evolutionary boundaries.

"Comparing ancient with modern populations of bacillus," he added,"we find the former out at the edges of the species distributionboundary."

Besides the dormancy seminar, Cano also manned a poster at themeeting, titled: "Characterization of ancient Bacillus thuringiensis-like isolates from amber, and their use in calibrating the 16S rRNAmolecular clock."

Ribosomal RNA's 16S component, Battista pointed out, "is a highlyconserved nucleic acid sequence. By comparing the differencesbetween two such sequences, you can gauge how old an organism is."

He continued: "We find similar nucleic acid sequences in a variety oforganisms. If you start looking at the molecular level in humans,bacteria, mice, you name it, what you will find are sequencesencoding proteins that are conserved _ that is, very similar to oneanother _ and used in all these different species.

"From that point of view, it certainly would imply that we're allessentially related, in the sense that there are certain highly conservedmolecules that we all use. And this would imply a radiating ofevolution from a common progenitor organism or organisms to thediversity of life on the planet."

Mutations Add Up To New Species

Battista outlined the inner works of the molecular clock, whichCano's Rip-Van-Winkle bugs help to calibrate:

"There is a concept," he said for starters, "that DNA is subject to asort of fixed mutation rate, and that as each bacterium divides there isa finite probability that its sequence will change. Estimates run in theneighborhood of one in 100 million chances, so since the organism'saverage genome is four million base pairs, it would have to gothrough two or three generations to accumulate a single mutation."

Every such change, he suggested, is a tick on the molecular clock."You can determine the sequence differences between ancient andmodern, and then infer how long ago the two organisms must haveseparated."

Cano observed: "We didn't know whether the clock exists at all, orwhether it ticks at a constant rate. That's one of the questions that wewanted to ask. And when we compared the number of basesubstitutions with modern species we found that their number wasconsistent with a clock.

"Our calculations or estimations of the clock were comparable tothose made by other scientists using other methods, all involving onlymodern organisms."

But that clockwork scenario presupposes a static planet.

"You have to correlate it to ecological events," Cano pointed out,"such as the appearance of oxygen in the earth's atmosphere, andstuff like that."

Just so, Battista concurred. "If there were some unanticipated,external influence that we do not understand, which accelerated orslowed down the spontaneous mutation rate in previous time, thenour molecular clocks would be off."

Molecular clock-watchers, both scientists agree, are "evolutionarybiology people."

Battista concluded: "Looking at a piece of amber 65 million yearsold, and making estimates that this animal, plant or microorganismwas there at that time, may assist us in adjusting the clock. Thiswhole idea of a molecular clock takes one aspect of evolution _ inthis case, bacteria _ and fits it better to the entire time line." n

(c) 1997 American Health Consultants. All rights reserved.